The camera used in a fluorescence microscope is arguably the most important component, and it can also be the most expensive. As a result, choosing the correct camera for your system is an important decision that should not be taken lightly.
We spoke to Ian Holland, from LUMENERA, about the different factors to consider when looking for a fluorescence microscopy camera, and the range of microscopy cameras that LUMENERA supply.
Why is it important to make an informed choice when choosing a new camera for your fluorescence microscope?
It is important to select the proper camera for fluorescence microscopy which has high sensitivity and low noise, to avoid photo bleaching your sample. A monochrome camera is also preferred for fluorescence imaging because it does not have a color filter pattern superimposed over the image sensor. Since the microscope’s filter wheel selects which wavelength makes its way to the sensor, a monochrome camera allows equal signal detection on all pixels and increases the overall sensitivity of the camera.
Color information is then added by software during image capture to match what is seen in the microscope’s eyepiece.
Every lab is budget-conscious these days, so selecting a camera that meets your needs is important. Choose a dedicated, high-end camera, if your application requires it. However, in other circumstances a single camera can perform well in both a brightfield and a fluorescence environment and cover the tasks typically requiring two cameras – saving money in the process.
Image taken using a Lumenera INFINITY3S-1UR Microscope Camera
What is the QE and peak QE? Are these the most important parameters to consider when choosing a fluorescence camera?
Quantum efficiency (QR) expresses the conversion ratio between photons of a given wavelength and electrons being freed on the image sensor to produce a signal. The higher the QE, the less light is required to fall on each pixel to obtain a usable signal. Peak QE denotes the wavelength at which the camera is best suited to convert photons into electrons. The camera’s overall QE graph indicates the camera’s QE in the range of each dye’s emission wavelength showing whether adequate conversion efficiency exists.
It is also important to note that some vendors express their camera’s QE in terms of relative QE instead of absolute QE. This normalizes the QE curve based on the camera’s peak QE, which would be set to 100%. These graphs are not adequate for camera comparison as only absolute QE can be used to differentiate cameras.
How can camera noise affect fluorescence measurements? Is camera cooling still necessary to successfully capture fluorescence images?
Cameras with both low read noise and low dark current noise are highly sought after in fluorescence microscopy because they produce clearer images and can benefit from the use of higher gain. Read noise occurs every time an image is captured. The lower the read noise, the higher the gain that can be used without deteriorating the image quality. This allows for shorter exposure times which helps to limit dark current noise. The latter form of noise is temperature and time dependent – typically expressed in electrons per second at a given temperature.
The shorter the exposure, the less dark current noise will be present in the image. With advancements in image sensor technology, noise levels have been on the decline by an order of magnitude over the last decade. This greatly reduces the need to cool cameras for most fluorescence applications and allows for significant cost savings for imaging equipment.
Do different types of fluorescence microscopy techniques require different camera specifications?
Many different types of research involve a variety of traditional and newer techniques using fluorescence microscopy. The strength of the emission signal from the sample and how well it endures the excitation energy are the main criteria impacting the sensitivity of the camera being selected.
Also, it is important to know that the camera sensor and/or filters in the optical path are compatible with the emission wavelength from the dyes being used, specifically if the emission lies beyond the visible spectrum (e.g. Near-IR).
Image taken using a Lumenera INFINITY3S-1UR Microscope Camera
How would the requirements for a camera used in multiphoton fluorescence imaging differ?
Multiphoton fluorescence imaging will generate emissions signals in the visible and Near-IR spectrum. So, the camera requirements will be similar for single photon or multiphon fluorescence. The camera selection will still depend on the strength of the signal from the specific dyes, their emission wavelength, and the power of the illumination source.
What problems are associated with using a substandard camera for fluorescence microscopy?
Selecting a microscope camera that is not intended for fluorescence microscopy will introduce significant image quality degradation. The image will either be too noisy to make out finer details in the intricate areas of the sample structure or too long an exposure time will be required to capture enough light, making it difficult to focus and position the sample in the field of view due to the extended time interval between images caused by long exposure times.
Also, many fluorescent dyes will have reduced emissions (photo-bleaching) the longer they are exposed to the light source, and a more light-sensitive camera allows for shorter exposure times, helping to minimize photo-bleaching.
What cameras do Lumenera provide for fluorescence microscopy?
Currently, the Lumenera INFINITY-3 series cameras are best suited for fluorescence applications, specifically the INFINITY-3S -1, our most sensitive and lowest noise camera in our portfolio.
The INFINITY-3-3URC and INFINITY-3-6URC camera models have excellent color reproduction for wide field imaging, while also being sufficiently sensitive to be used for bright fluorescence and allowing a single camera to perform the job typically requiring two separate cameras.
Does Lumenera provide assistance for customers in deciding which camera is best for their application?
Lumenera support is there for you every step of the way, from selecting the right camera for your application and budget to phone and e-mail support if questions arise after the purchase of your camera through our Technical Assistance Center (TAC). Technical support is included in the purchase price of the camera and does not have a time or usage limit.
Does Lumenera have any other advice on designing a robust fluorescence microscopy system?
Selecting the right camera for your application can pay back in dividends down the road. Service, build quality and usability go a long way in ensuring ease of use, product longevity, and reducing downtime in the lab. Lumenera offers an industry-leading four-year warranty on all of our cameras.
We stand behind our products and are committed to providing our customers with meaningful support throughout the life of their camera.
Where can our readers find out more about Lumenera and the cameras you provide for fluorescence microscopy?
Our website has a complete list of our microscopy cameras that are currently available. And, we can be reached with questions at [email protected].
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About Ian Holland
Since 2005, Ian’s role at Lumenera has been centered around the scientific customer by helping them select which cameras are most suitable for their applications, including digital pathology. He also offers global customer and sales support as part of the TAC team.
As a Senior Applications Engineer, Ian is a product manager for the INFINITY ANALYZE software. His entire career has been focused in customer support and service and has been in the image processing industry since 1985.